Systems and methods for continuous flow digital droplet polymerase chain reaction bioanalysis

ABSTRACT

Systems and methods for continuous flow polymerase chain reaction (PCR) are provided. The system comprises an injector, a mixer, a coalescer, a droplet generator, a detector, a digital PCR system, and a controller. The injector takes in a sample, partitions the sample into sample aliquots with the help of an immiscible oil phase, dispenses waste, and sends the sample aliquot to the mixer. The mixer mixes the sample aliquot with a PCR master mix and diluting water, dispenses waste, and sends the sample mixture (separated by an immiscible oil) to the coalescer. The coalescer coalesces the sample mixture with primers dispensed from a cassette, dispenses waste, and sends the reaction mixture (separated by an immiscible oil) to the droplet generator. The droplet generator converts the sample mixture into an emulsion where aqueous droplets of the reaction mixture are maintained inside of an immiscible oil phase and dispenses droplets to the digital PCR system. The digital PCR system amplifies target DNAs in the droplets. The detector detects target DNAs in the droplets. The controller controls the system to run automatically and continuously.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of U.S. Prov. Pat. App. Ser.No. 62/186,321, having the same title and filed Jun. 29, 2015, andincorporated fully herein by reference.

BACKGROUND

The technology of polymerase chain reaction has been a common and oftenindispensable technique in medical and biological studies andapplications. Digital PCR (dPCR) allows quantification of DNA in asample. dPCR is advantageous for reasons of accuracy (absolute titerquantification), sensitivity (single molecule detection), dynamic range,and robustness against inhibition. A mobile dPCR allows immediatequantification of samples, but the samples typically need to be purifiedbefore a dPCR can be conducted. In addition, a vast amount of samplesmay need to be tested, compared to typical lab settings.

Systems and methods of a portable continuous flow dPCR device thatautomates the entire analysis on a continuous flow of samples from afluid are described herein.

SUMMARY

The present disclosure provides systems and methods that perform digitaldroplet PCR analysis on a continuous fluid stream. The instrument drawsin a sample of molecules, such as DNA in aqueous suspension, mixes anddilutes that sample with PCR mastermix, a diluent such as water, and oneor more suitable PCR probes without disrupting flow of the fluid streamsignificantly. The resultant sample liquid is then broken into dropletsthat stochastically contain the target molecules. The droplets are thenthermocycled to amplify their nucleic acid contents by PCR. In the end,the individual droplets are counted to determine the original startingconcentration in the sample.

In accordance with one aspect of the disclosure, a system for continuousflow polymerase chain reaction (PCR) is provided. The system comprisesan injector, a mixer a droplet generator, a detector, a digital PCRsystem, and a controller. The injector takes in a sample from a sampleinlet and aliquots the sample into a volume necessary for a PCRreaction, dispenses waste, and hands off the sample aliquots separatedby an immiscible oil phase to a mixer one aliquot at a time. The mixertakes in the sample aliquot, mixes it with the PCR master mix anddiluting water, dispenses waste, and hands off the sample mixture to acoalescer in aliquots separated by an immiscible oil phase. Thecoalescer takes in the sample mixture, coalesces it with primers thatare dispensed from the cassette, dispenses waste, and hands off thereaction mixture separated by an immiscible oil phase to the dropletgenerator. The droplet generator converts the sample mixture into anemulsion where aqueous droplets of the reaction mixture are maintainedinside of an immiscible oil phase. The aqueous reaction droplets arethen passed to the digital PCR system to enable amplification of targetmolecule (e.g., DNA) molecules in the droplets. Post amplification, adetector determines whether or not target molecule (e.g., DNA)amplification occurred for each of the droplets. The controllerprocesses data outputted from the detector and controls the system sothat the system runs automatically and continuously.

In another aspect of this disclosure, a method for continuous flow PCRis provided. First a sample of a fluid stream is taken in at a sampleinlet and passed through an injector to produce sample aliquots, witheach aliquot being separated by an immiscible oil phase. Each samplealiquot is mixed, e.g., using a mixer, with reagents such as PCR mastermix, primers, probes, and diluting water to produce a sample mixture.The primers and/or probes may be PCR primers modified with fluorophoresthat bind to a target molecule, such as DNA. The reagents may come froma cassette or from reagent storage.

The foregoing and other advantages of the invention will appear from thefollowing description. In the description, reference is made to theaccompanying drawings, which form a part hereof, and in which there isshown by way of illustration a preferred embodiment of the invention.Such embodiment does not necessarily represent the full scope of theinvention, however, and reference is made therefore to the claims andherein for interpreting the scope of the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram of digital polymerase chain reaction(PCR).

FIG. 2 is a photo of an example system implemented according to thepresent application.

FIG. 3 is a schematic diagram of an example system implemented accordingto the present application.

FIG. 4 is an example flowchart illustrating a method implementedaccording to the present application.

FIG. 5 is an example output from the photomultiplier tube LEDs.

FIG. 6 is an example plot of percent positive droplets versus the numberof copies of the DNA.

FIGS. 7(A)-(B) show schematics depicting an example thermocycler.

FIGS. 8(A)-(D) show schematics of the wraps of the heater cores in anexample thermocycler.

FIG. 9 shows an example interweaving mechanism of wraps in the heatercores of an example thermocycler.

FIG. 9A shows a schematic diagram of the multiple inlet ports of anexample injector.

FIG. 10 show the structure of an example coalescing element.

DETAILED DESCRIPTION

“Polymerase chain reaction” or “PCR” refers to a technology widely usedin molecular biology to amplify a single copy or a few copies of DNAacross several orders of magnitude, generating thousands to millions ofcopies of a particular DNA sequence.

The PCR technology uses reaction mixture that comprises DNA templatescontaining DNA to be amplified, primers, enzyme such as Taq polymerase,deoxynucleoside triphosphates (dNTPs)—the building-blocks from which theDNA polymerase synthesizes a new DNA strand, buffer that provides asuitable chemical environment for the amplifying process, and otherchemicals. PCR master mix comprises those components except primers.Primers are short DNA fragments containing sequences complementary tothe target region along with a DNA polymerase are used to enableselective and repeated amplification. As PCR progresses, the DNAgenerated is itself used as a template for replication, setting inmotion a chain reaction in which the DNA template is exponentiallyamplified.

The PCR methods comprises placing the reaction mixture in a thermocyclerand, in the thermocycler, undergoing a series of 20-40 repeatedtemperature changes—called cycles—with each cycle commonly consisting of2-3 discrete temperature steps. The cycling is often preceded by asingle temperature step at a high temperature (>90° C.)—also called hotstart, and followed by one hold at the end for final product extensionor brief storage. The temperatures used and the length of time in eachcycle depend on parameters, such as the enzyme used for DNA synthesis,the concentration of divalent ions and dNTPs in the reaction, and themelting temperature of the primers.

Each cycle usually comprises three steps, melting (or denaturation),annealing, and extension (or elongation). In the melting step, thereaction mixture is heated to 94-98° C. for 20-30 seconds, causingmelting of the DNA template to single-stranded DNA molecules bydisrupting the hydrogen bonds between complementary bases.

In the annealing step, the reaction temperature is lowered to 50-65° C.for 20-40 seconds allowing annealing—combining—of the primers to thesingle-stranded DNA template. This temperature is low enough to allowfor hybridization of the primer to the strand, but high enough for thehybridization to be specific, i.e., the primer should only bind to aperfectly complementary part of the template. Stable DNA-DNA hydrogenbonds are only formed when the primer sequence very closely matches thetemplate sequence. The polymerase binds to the primer-template hybridand begins DNA formation.

In the extension step, the DNA polymerase synthesizes a new DNA strandcomplementary to the DNA template strand by adding dNTPs that arecomplementary to the template.

Digital PCR follows the same principle and process as those oftraditional PCR, except that, in digital PCR, a sample is partitionedinto many small partitions such that individual nucleic acid templatesof interest can be localized in individual partitions.

Referring to FIG. 1, a schematic illustrating digital PCR is provided.In step 102, bulk sample is placed in tube 110. The bulk sample containsmany nucleic acids or DNA, as shown in the insert 112. In step 104, thesample is partitioned into many individual reactors 114. Each of thereactors may or may not contain a target DNA. In step 106, each reactorundergoes a PCR such that the number of target DNAs in a reactor isamplified to a detectable level. In step 108, the partitioned sample isdigitally read out, providing quantification.

Referring to FIG. 2, a photo of an example system implemented accordingto the present application is provided. The system can be enclosed in abrief case 202 equipped with external power outlet. The system providesa sample injection port 206, a reagent bay 208, and a primer library210. A controller 204 controls the system and analyzes data. Thecontroller can be a tablet PC (as shown in FIG. 2), a laptop, or amobile device.

Referring to FIG. 3, a schematic illustrating an example system 300implemented according to the present application is provided. The systemcomprises an injector 302, a mixer 304, a coalescer 306, a dropletgenerator 308, a digital PCR system, a detector, and a controller 204.The digital PCR system comprises a thermocyler 310. The detector cancomprise a droplet counter 312. The sample injection port 206 providesan inlet for inputting a sample into the injector 302. The reagent bay208 holds reagents to be mixed with the sample in the mixer 304. Theprimer library 210 holds primers used to detect the target DNA. Theprimers from the primer library 210 may be fed to a cassette 314, or thecassette 314 may include the primer library 210. The cassette 314 handsoff the primers to the coalescer 306. Then the primers are coalescedwith sample mixture in coalescer. The controller 204 controls the systemand analyzes the data detected by the detector. As shown in FIG. 3, inthe system 300, waste outlets are available at each major step and oilcan be used throughout to act as a carrier fluid for the sample. Theinjector 302, mixer 304, and coalescer 306 can automatically hand offthe sample mixture to the next unit so that the sample mixture flowsthrough the system 300 continuously.

Referring to FIG. 4, a flowchart 400 depicting an example methodimplemented according to the present application is provided. At step402, the sample is partitioned with oil in an injector 302. The sampleis inputted into the system through a sample injection port 206. Wasteis dispensed after the mixing and the ejector 302 hands off the samplemixture to the mixer 304. In step 404, the sample mixture handed offfrom the injector is mixed in the mixer 304 with the PCR master mix anddiluting water. The master mix is held in reagent bay 208. Again, wasteis dispensed and the mixer 304 hands the sample mixture off to thecoalescer 306. In step 406, the sample mixture is coalesced with primersinto reaction mixtures. Fluorescent-labeled primers can be used todetect target DNAs. Also, waste is dispensed. In step 408, the reactionmixture is broken up into droplets using the droplet generator oil andthe droplets are dispensed by the droplet generator 308. The dropletscan be used in a droplet digital PCR system. For example, the target DNAis amplified with the temperatures cycled and controlled by thermocycler310. Before the temperature cycles, droplets may go through a hot startstep. After the target DNA in the droplets are amplified, theconcentration of target DNA in the sample can be detected by countingfluorescent-labeled droplets detectable by photo-multiplier tube LEDs inthe mixture of the droplets and oil. Waste is dispensed.

Referring to FIG. 5, an example output from the PMT-LEDs is provided.Each peak marked with a circle denotes the signal of a droplet detectedby the PMT-LEDs. When a droplet has target DNA, the target DNA in thedroplet is fluorescent labeled due to the fluorescent-labeled primers.When such a droplet passes through the droplet counter 312, the signalstrength is higher than that of a droplet without the target DNA. Athreshold 504 can be set to count the number of droplets having thetarget DNAs.

Referring to FIG. 6, the amount of a target sequence or gene that ispresent in the plurality of droplets measured by Applicants' apparatusis comparable to the amount of the target sequence or gene measured byreal-time PCR, also called quantitative PCR (qPCR). A series ofdilutions of the target sequence or gene is prepared and indicated bythe series of copy numbers of the x-axis. C_(t) (threshold cycle) isemployed here to quantify the relative measure of the concentration ofthe target sequence or gene. The red dot indicates a measurement of thetarget sequence or gene at a different dilution of the sample measuredby the current apparatus. FIG. 6 shows that the red dots are close tothe predicted concentrations of the target sequence or gene at differentdilutions of the sample and are well within the range of minus or plus0.5 C_(t) value of the concentrations of the target sequence or gene.

Specifically for real-time PCR, the thermocycler must have the abilityto maintain a consistent temperature, as PCR amplification efficiency isdependent upon the temperature. Referring to FIGS. 7(A)-(B), schematicsdepicting an example thermocycler are provided. The thermocycler 310comprises functioning components—heater cores 702 (in FIG. 7(A)). Theheater cores 702 is covered with foam caps 704 to insulate and providestructural support for the heater cores 702. Together with the foam caps704, the heater cores 702 are placed in a housing 706 that seals andinsulates the cores 702. The temperature of the cores 702 are controlledby a circuit board 708 using the temperature as feedback.

Referring to FIGS. 8(A)-(D), schematics of the wiring of the heatercores of an example thermocycler are provided. The heater cores 702comprises tubings and wraps around the tubings. The heater cores cancomprise two tubings 804 and 810. For example, tubing 804 can be a 95°C. tubing having 40 continuous hot start wraps 802, and tubing 810 can a60° C. tubing. Wraps for hot start are wrapped around one tubing 804 (inFIG. 8(B)) and thermocycle wraps 806 and 808 are wrapped around theother tubing 810 or both tubings (wraps 806 wrap around tubing 810 andwraps 808 wrap around both tubings 804 and 810, shown in FIG. 8(A)-(C)).As such, the hot start and thermocycle wraps are interweaved aroundtubing 804 as shown in FIGS. 8(A) and (D). The detail of an exampleinterweaving mechanism is shown in FIG. 9. The thermocycle wraps 806 canflow downward. Two thermocycle wraps per cycle can be used to extendanneal time. Two wraps 808 of tubing 810 (e.g., a 60° C. tubing) caninterweave with incoming wraps 802 around tubing 804 (e.g., a 95° C.tubing) to balance heat load distribution. When the two sets of wrapsinterweave, hot start wraps 802 flow in the opposite direction ofthermocycle wraps 808 to balance heat load distribution (e.g., hot startwraps 802 flow upward with individual 95° C. thermocycle wraps 808flowing downward). The wrap arrangement as disclosed herein can maintainmore constant temperature at each point on the core, so the powerconsumption is lower for heating and cooling and, in turn, this moreconsistent temperature gives better results to the PCR reaction.

The injector 302 can have multiple ports of different specific volumes(as shown in FIG. 9A, ports include sizes 2.5, 5, 25, and 50microliters) and low dead volumes. The connections of the injectors canbe Teflon or fluoroplastic. Such materials, like Teflon andfluoroplastic, have low surface energy and do not contaminate thesample. Because of the low surface energy, cleaning solutions do notabsorb into the connections; thus, the injectors are bleach cleaningcompatible and the connections can be reused.

Referring to FIG. 10, to combine all of the inserted reagents into asingle reagent outlet a component may include outlet stator 1002, rotorwith reagent chamber 1004, inlet stator 1006, and position encoder 1008.The stator can be made of Teflon. The reagent chamber can be initiallyfilled with oil, such as fluorocarbon oil, to be used as carrier. Therotor 1004 may be rotated to fill reagent storage chamber with variousreagents. Then high voltage field is used to induce electrocoalescenceof all reagents. Afterwards, reagents and unneeded oil are flushed aswaste through outlet to downstream processes. The fluid connections canbe all Teflon or fluoroplastic. A vertical orientation with 60° coneangle may be used to allow for sample outlet at up to 45° tilt duringoperation. Arbitrary volumes may be passed at arbitrary flow rates up tocapacity of the chamber 1004. Buoyancy of reagents relative to fluiddrives reagent close to packing. A rotor 1004 edge can automaticallycleave inlet reagent to a specified volume. As the rotor 1004 turns, itcuts a cylindrical slug of reagent into smaller volumes of known volume.

The present invention has been described in terms of one or morepreferred embodiments, and it should be appreciated that manyequivalents, alternatives, variations, and modifications, aside fromthose expressly stated, are possible and within the scope of theinvention. The appended document describes additional features of thepresent invention and is incorporated herein in its entirety byreference.

We claim:
 1. A sample analyzing system, comprising: an injector thatreceives a sample, the injector comprising an injector inlet and aninjector outlet; at least one mixer valve in fluid communication withthe injector, the mixer receiving the sample from the injector andmixing the sample with a plurality of polymerase chain reaction (PCR)reagents to produce a reagent composition; a cassette in fluidcommunication with the at least one mixer and storing the plurality ofPCR reagents including master mix, primers, probes, and diluent; adroplet generator receiving the reagent composition, the dropletgenerator comprising a generator inlet and a generator outlet fordispensing a plurality of droplets of the mixture; a thermocycler influid communication with the droplet generator and receiving theplurality of droplets of the mixture and performing PCR on the pluralityof droplets; a fluorescence detecting apparatus in fluid communicationwith the thermocycler for quantifying at least one molecular target indroplets that contain the at least one molecular target; and acontroller comprising: a processor in signal communication with each ofthe injector, the mixer, the cassette, the droplet generator, thethermocycler, and the fluorescence detecting apparatus; and anon-transitory computer readable medium comprising program instructionsexecutable by the processor to control the injector, the mixer, thecassette, the droplet generator, the thermocycler, and the fluorescencedetecting apparatus.
 2. The system of claim 1, wherein the reagentcomposition is produced by electrocoalescence of the sample with theplurality of PCR reagents.
 3. The system of claim 2, further comprising:an inlet stator having a first plurality of apertures extendingtherethrough; an outlet stator having a second plurality of aperturesextending therethrough; a rotor having a third plurality of aperturesextending therethrough and an internal cavity, the rotor disposedbetween the inlet stator and the outlet stator; and a reagent chamberdisposed within the internal cavity of the rotor, wherein the reagentchamber is configured to rotate along an edge of the rotor from oneaperture to another, and wherein at least a portion of the reagentcomposition is produced by electrocoalescense within the reagentchamber.
 4. The system of claim 3, wherein the reagent chambercomprises: a tubular container; a cone connecting to a first end of thecontainer; and a receiver connecting to an apex of the cone and having afirst side for receiving the plurality of PCR reagents and a second sidefor dispensing the reagent composition.
 5. The system of claim 1,wherein the cassette comprises a housing and a at least one chamber,wherein the chamber and the chamber stores the plurality of PCRreagents.
 6. The system of claim 1, wherein the droplet generatorcomprises a fluorophilic surface.
 7. The system of claim 1, wherein thedroplet generator comprises a hydrophobic surface.
 8. The system ofclaim 1, wherein the droplet generator comprises a fluorophilic andhydrophobic surface.
 9. The system of claim 1, wherein the thermocyclercomprises: a plurality of heaters; a plurality of insulating blocksconfigured to support and insulate the plurality of heaters; and aplurality of wraps configured to wrap around the plurality of heatersand carry the fluid stream around the plurality of heaters.
 10. A methodfor analyzing a sample, the method comprising the steps of: receivingthe sample in an injector; partitioning the sample with a firstimmiscible oil to obtain a sample aliquot; delivering the sample aliquotto a mixer; mixing the sample aliquot with a first plurality of PCRreagents to obtain a sample mixture; mixing the sample mixture with asecond plurality of PCR primers for performing PCR to obtain a reactionmixture; generating a plurality of droplets of the reaction mixture; andquantifying at least one molecular target contained in the plurality ofdroplets of the reaction mixture.
 11. The method of claim 10, whereinreceiving the sample further comprises lysing the sample and isolating aplurality of nucleic acids.
 12. The method of claim 11, whereinisolating the plurality of nucleic acids comprises isolatingdeoxyribonucleic acid (DNA) of the sample.
 13. The method of claim 11,wherein isolating the plurality of nucleic acids comprises isolatingribonucleic acid (RNA) of the sample.
 14. The method of claim 10,wherein partitioning the sample with the first immiscible oil to obtainthe sample aliquot further comprises dispensing a first waste.
 15. Themethod of claim 10, wherein mixing the sample aliquot with the firstplurality of PCR reagents to obtain the sample mixture further comprisesdispensing a second waste.
 16. The method of claim 10, wherein mixingthe sample mixture with the second plurality of PCR primers to obtainthe reaction mixture further comprises dispensing a third waste.
 17. Themethod of claim 10, wherein generating the plurality of droplets of thereaction mixture comprises using high voltage electrocoalescence togenerate the plurality of droplets of the reaction mixture.
 18. Themethod of claim 10, wherein mixing the sample mixture with the secondplurality of PCR primers comprises mixing the sample mixture with aplurality of fluorescently labeled PCR primers.
 19. The method of claim10, wherein quantifying the at least one molecular target contained inthe plurality of droplets of the reaction mixture comprises measuringfluorescent strength of a fluorescent labeled for at least one moleculartarget.